Method of using manganese to protect an animal from radiation damage

ABSTRACT

An antioxidative biophylactic agent containing a substance that can induce metallothionein production in human or animal body. Manganese or compounds thereof, Streptococcus hemolyticus which has been deprived of a streptolysin-S-yielding ability, and an extract from coriolus versicolor belonging to the family Polyporaceae, Basidomycetes having a remarkable metallothinonein-inducing ability in vivo, and an antioxidative biophylactic effect such as radiation damage protection can be obtained by administering these metallothionein-inducing substances.

TECHNICAL FIELD

This invention relates to a pharmaceutical agent acting on theprotection mechanisms in human or animal bodies and, more particularly,to a pharmaceutical agent having activity of protecting living bodiesfrom non-specific oxidative invasions thereof.

BACKGROUND OF THE INVENTION

Living bodies are exposed to a variety of environmental impacts. Suchimpacts are almost unlimited in number, including not only physicalstimulations such as unceasingly arriving cosmic rays, radiations fromthe ground surface or buildings, ultraviolet rays in the sunlight andchanges in ambient temperature but also chemical stimulations due toabsorption of trace polutants in the air or foreign substances in foodsand biological invasions by invisible viruses, bacterial, fungi, etc.Each living body is endowed with a very sophisticated and potentprotection mechanism and it is this protection mechanism that allow theliving organism to survive while overcoming those variegated invasions.

Fundamentally, the mechanism of living body damage by oxidativeinvasions from the environment and the defense mechanism are common inmany aspects irrespective of differences in environmental factors. Thus,it is known that radiation damages, oxidant damages, inflammations,immunization, aging, carcinogenesis and various enzymatic processes areassociated with free radical formation or active oxygen. It is alsoknown that when many drugs act in human bodies, their metabolicprocesses involve free radical formation as a part thereof. Free radicalformation is generally a factor harmful to healthy tissues except forits usefulness in some cases, for example in the case of microbicidalaction of leukocytes or activation of certain enzymes in which .O₂ ⁻(superoxide radical) is physiologically involved. Living bodies have anonspecific defense system against such free radical formation. More indetail, when a living body is exposed to a radiation or oxidant or to anaction of a drug, such agent not only directly damages substances ofbiological importance, such as DNA, but also reacts with O₂ in the airto produce .O₂ ⁻ or reacts with water (H₂ O) in a tissue to formstrongly reactive radicals such as .OH (hydroxy radical). The .O₂ ⁻radical is partly eliminated as a result of conversion to H₂ O₂ by SOD(superoxide dismutase) occurring in tissues. While catalase converts H₂O₂ to water and oxygen, H₂ O₂ also reacts with .O₂ ⁻ to form .OH, whichhas very high reactivity and attacks a variety of living-bodysubstances. In particular, these reactions, via formation of lipidradicals, lead to peroxidation of unsaturated fatty acid components ofphospholipids which are constituents of biomembranes. It is vitamin Ethat plays a definite role in blocking chain reactions involving thosefree radicals that are formed from such membrane lipids. When subjectedto lipid peroxidation, biomembranes undergo polymerization and theirfunctions are thereby disturbed. This results in temporary inhibition ofdifferent biochemical reactions or in abnormalities in theadrenocortical hormone activities. Damages of biomembranes due to theformation of lipid peroxides are prevented by the peroxide eliminatingaction of the glutathione (GSH)-peroxidase system (GP system).

As mentioned above, living organisms are subjected to various oxidativeinvasions from outside and inside. At the same time, however, thedefense mechanism functions in them through the action of vitamin E andthe radical- or peroxide-eliminating action of the above-mentioned SODor GP system, among others. In case this defense mechanism fails tofunction satisfactorily, long-term effects of said invasions will resultin such general phenomena as ischemic disorders of the brain or heart,damages of the lung and aging of cells. Such various radicals may damageDNA molecules directly and this may lead to carcinogenesis. Furthermore,a number of chemical carcinogens and intermediate metabolites includefree radicals themselves or can activate free radicals. Thus, theproblem of defense against free radicals and various disordersassociated therewith is a problem of protection against carcinogenesisas well.

PRIOR ART

Any drug capable of effectively inhibiting and eliminating free radicalshave not been developed as yet. The activity of vitamin E which is saidto inhibit the formation of lipid radicals is not yet stable ineffectiveness when evaluated from the system effect viewpoint. Thus,even a substance which has an effective activity on the cellular levelcannot be used as a drug if it shows a severe side effect which maydisturb the systemic balance. Generally drugs have to be used at doseslower than the doses at which their side effects pose a problem.

In cancer treatment, irradiation is generally used for the purpose ofdestroying cancer cell nuclei. Under the present conditions, however,few measures are taken against associated systemic adverse effects dueto irradiation on other sites than the target sites.

In therapy and various industries dealing with radiations, a variety ofmeans are taken to prevent workers from exotic exposure to radiations.However, any drugs effective in preventing radiation damages or reducingdamages without causing adverse effects in case of exposure have notbeen developed as yet. Although cysteamine, S,2-aminoethylisothiuroniumand S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721), amongothers, are known as sulfur-containing substances having an SH groupwhich can function as protective substances against radiations and theyare highly effective in preventing radiation damages in bone marrow stemcells, they are cytotoxic and produce very severe side effects. Themargin between the toxic dose and effective dose is little. Therefore,they are hardly applicable to human bodies. As for glutathione which iseffective in physiologically protecting organisms from radiations, itseffect can be expected only at large doses since its entering cells isdifficult and the retention time in the body is short (half-life: 2minutes) even when it is administered from the outside.

As mentioned above, the defense mechanism in living bodies in which theabove-mentioned SOD and GP systems play principal roles is the onlymeasure relied upon in coping with disorders or damages of living bodiesas resulting from free radical formation. At present, no particularmeans of actuating said mechanism of systematic viewpoint is employableas yet. However, when abnormal free radical formation is induced in thebody, as in the case of irradiation particularly for cancer treatment,the defense mechanism which the living body itself has is no moresufficient but allows occurrence of various functional disorders such asmentioned above.

In view of such situation of the prior art, it is an object of theinvention to provide a pharmaceutical agent capable of stimulatingbody's protection mechanism and inhibiting damages of living bodies ascaused by various oxidative invasions such as exposure to radiations.

DISCLOSURE OF THE INVENTION

As a result of her intensive investigations to achieve the above object,the inventor found that metallothioneins (hereinafter referred to asMTs), which are metalloproteins, can be an alternative of glutathionewhich contribute to the inhibition of functional disorders due tobiomembrane peroxidation and so forth, and that an antioxidativebiophylaxis-potentiating activity, such as a protective activity againstradiation damages, can be developed when MTs are induced in livingorganisms. The present invention has now been completed based on thesefindings.

Thus, the invention consists in an antioxidative biophylactic agentcharacterized in that it contains a substance capable of inducing MTs inliving human or animal bodies.

MTs are low-molecular proteins having molecular weight of 6,000-7,000and very rich in SH groups due to cysteine accounting for about onethird of the constituent cells amino acids. They are known to be presentin living body of various kinds from microorganisms to higher organisms.MTs contribute to alleviation of symptoms of intoxication from heavymetals such as Cd and Hg and estimatedly also play an important role inhomeostasis of the concentration of Zn, which is one of essentialelements, although their physiological activities have not yet beenfully elucidated [M. Webb et al.: Biochem. Pharmacol., 31, 137 (1982);A. Karin: Cell, 41, 9 (1985)]. Furthermore, it is known thatadministration of Cd, Zn, Cu, Hg, Au and Ag induces MTs in tissues ofliving organisms ["Seitai to Jukinzoku (Living Organisms and HeavyMetals)", written and edited by Keiichiro Fuwa, published by Kodansha(1981)]. However, that MTs induced by a substance administered from theoutside exhibit a protecting action against oxidative invasions inliving organisms and that remarkable protective effects can be obtainedby administration of the substances mentioned below are the findingobtained for the first time by the inventor.

Thus, those metals so far known to be capable of inducing MTs, such ascadmium, zinc, copper, mercury, gold and silver, or compounds of suchmetals can be used as the substance capable of inducing MTs in thepractice of the invention. The inventor found that manganese andcompounds thereof, hemolytic streptococci (Streptococcus haemolyticus)treated for elimination of the streptolysin S production capacity(hereinafter referred to simply as hemolytic streptococci), and anextract of Coriolus versicolor Quel, which belongs to the orderPolyporales of the class Basidiomycetes, have marked MT-inducingcapacity and exhibit marked antioxidative and biophylaxis-potentiatingactivity, for example protective activity against radiation damages.

In the case of application to human bodies, manganese, zinc and gold andcompounds of these, the above-mentioned hemolytic streptococci and theabove-mentioned Coriolus versicolor extract are preferable from thetoxicity viewpoint, and manganese and compounds thereof, theabove-mentioned hemolytic streptococci and the above-mentioned Coriolusversicolor extract are preferable particularly from the efficacyviewpoint. As manganese, water-soluble manganese salts are morepreferably used.

Hemolytic streptococci constitute a class of streptococci capable ofproducing streptolysin, a hemolytic toxin, and are pathogenic bacteriacausing erysipelas, septicemia, puerperal sepsis, tonsillitis andvarious other diseases. However, it is known that they can be applied tohuman bodies after elimination of their ability to produce streptolysinS which is stable against oxygen (Japanese Patent Laid-open PublicationNo. 56-79622).

In the practice of the invention, any strains of hemolytic streptococcican be used if the streptolysin S production capacity has beeneliminated. The term "hemolytic streptococci" as used herein includesvarious kinds of cells treated in an appropriate manner, such as thestrains Streptococcus pyogenes Su (ATCC 21060), Streptococcus pyogenesC203S (ATCC 21546), Streptococcus pyogenes S-43 (ATCC 21547),Streptococcus pyogenes Black-more (ATCC 21548) and Streptococcusequisimilis (ATCC 21597), each treated in various ways.

A typical method for the elimination of the streptolysin S productioncapacity of these bacterial cells comprises suspending viable cells in asalt solution, for example Bernheimer's basal medium (BBM), containingpenicillin at a relatively high concentration and maintaining thesuspension at 30°-38° C. [Jpn. J. Exp. Med., 36, 161-171 (1966)]. Amethod which comprises treating the suspension further at 40°-50° C.(Japanese Patent Publication No. 43-6690) can also be used. Furthermore,the use of cephalosporin C or cycloserine in lieu of penicillin givesequivalent results (British Patent No. 1,153,113; Japanese PatentPublications No. 45-8871 and No. 46-2674). In addition, preparationscontaining a smaller number of viable cells can be obtained by treatingwith hydrogen peroxide or a monohydric alcohol in the above process step(Japanese Patent Publication No. 55-43754; Japanese Patent Laid-OpenPublication No. 57-18621).

L-Group hemolytic streptococci which are obtainable by cultivating cellsof a hemolytic streptococcal strain under conditions such that the cellwall synthesis is inhibited, for example in a penicillin-containinghypertonic medium or by treating hemolytic streptococcal cells with abacteriolytic enzyme and then cultivating said cells in a mediumcontaining an inhibitor of cell wall synthesis such as penicillin areknown to be free of such drawbacks as phlegmogenicity, pyrogenicity andpain-causing property and can be used as preferable streptococcal cellsas well (Japanese Patent Laid-Open Publication No. 56-15210).

A particularly preferred hemolytic streptococcal preparation usable inthe practice of the invention is composed of cells of the attenuated,human-derived A hemolytic streptococcal strain Streptococcus pyogenes Suas treated with penicillin G potassium, and a cell preparation obtainedby lyophilizing said cells is known as OK-432. OK 432 is a lyophilizatederived from a treated culture product obtained by cultivating theabove-mentioned cells in Bernheimer's basal medium containing penicillinG potassium (not less than 25,000 units/ml, preferably 27,000-60,000units/ml) at 30°-38° C. (preferably 37° C.) for 10-30 minutes(preferably 20 minutes) and then incubating at 30°-50° C. (preferably45° C.) for 20-40 minutes (preferably 30 minutes), with hydrogenperoxide treatment being performed in the meanwhile and occurs as awhite to almost white, hygroscopic lyophilizate powder containing astabilizer. Its suspension formed upon addition of physiological salinehas a pH of 5.5-7.5, with an osmotic pressure ratio of 1 relative tophysiological saline. OK-432 shows no streptolysin S production capacitybut shows disappearance of the capsule and partial damage of the cellwall as compared with the starting material viable Su strain cells. TheLD₅₀ value in dogs is 36 KE/kg (1 KE corresponding to 0.1 mg oflyophilized cells). For the method of production, properties, biologicalcharacteristics, pharmaceutical characteristics such as toxicity, andphysiological activities of OK-432, refer to Nakao Ishida and TakashiHoshino: "Hemolytic Streptococcal Preparation OK-432", published byExcerpta Medica (1985).

The Coriolus versicolor extract can be used in the form of a fractionobtained, for example, by extracting mycelia of Coriolus versicolor Quelwith hot water, saturating the supernatant with ammonium sulfate,collecting the resultant precipitate and desalting the same. Saidfraction is known as PS-K [Shigeru Tsukagoshi: Gan to Kagaku Ryoho(Cancer and Chemotherapy), 1, 251-257 (1974); Igaku no Ayumi (Advaces inMedicine), 91, 505-510 (1974)]. This PS-K is a glycoprotein containing19 kinds of amino acids inclusive of aspartic acid and glutamic acidwith a protein content of about 15% and is known to have low toxicity.

The antioxidative biophylactic agent according to the invention isuseful typically as a protective agent against radiation damages due toradiations such as X rays, alpha rays, beta rays, gamma rays, neutronbeams, accelerated electron beams and ultraviolet rays. Theabove-mentioned hemolytic streptococci and Coriolus versicolor extractare known to have antitumor activity and, when they are used on theoccasion of radiotherapy for cancer treatment, they should preferablyhave a high degree of antitumor activity and, in this case they can besubjected to various treatments for increasing the antitumor activity inaddition to the above-mentioned treatments to increase more potentialityto induce MTs.

The antioxidative biophylactic agent according to the invention can beadministered either orally or non-orally in the form of theabove-mentioned substance capable of MT induction as it is or in dosageforms such as powders, granules, tablets, capsules or injectionsprepared by admixing said substance with pharmaceutically acceptablediluents, excipients, carriers and/or the like. The dose may varygreatly depending on the specific purpose of use as an antioxidativebiophylactic agent, the MT induction capacity of the above-mentionedsubstance, the target of administration, the route of administration,and other factors, hence cannot be particularly limited. Generally,however, it amounts to 0.1-1,000 mg/kg of body weight peradministration. In cases where it is used for preventing radiationdamages, it should desirably be administered about one day prior toirradiation. The protective effect of water-soluble manganese onradiation damages is remarkable and, in this case, a salt containing thedivalent Mn ion, such as manganese chloride or manganese sulfate, isdesirably administered at a dose of 10-30 mg of Mn per kilogram of bodyweight. The above-mentioned hemolytic streptococci and Coriolusversicolor extract also have remarkable protective activity againstradiation damages and, in this case, an optimum dose per administrationis roughly within the range of 0.1-10 mg/kg body weight in the case ofhemolytic streptococci and 10-1,000 mg/kg body weight in the case ofCoriolus versicolor extract.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of the cumulative mortalities in miceof metal-dosed and other groups after irradiation with 630 rad of Xrays.

FIG. 2 is a graphic representation of the changes in body weight in miceof Mn-dosed and other groups after irradiation of 630 rad of X rays inthe same test example.

FIG. 3 is a graphic representation of the survival rates in mice ofdifferent Mn-dosed groups after irradiation with 720 rad of X rays.

FIG. 4A and FIG. 4B are graphic representations of the relationshipbetween the survival rate in mice of each group after irradiation with630 rad of X rays and the dose of Mn and the relationship between saidsurvival rate and the logarithm of the liver MT concentration,respectively, each with a coefficient of correlation as determined bysimple regression analysis.

FIG. 5 is a graphic representation of the cumulative mortalities inrelation to the number of days after irradiation with gamma rays in micedrinking zinc-containing water.

FIG. 6 is a graphic representation of the survival rates in mice of anOK-432-dosed group after irradiation with 720 rad of X rays, which isthe lethal dose.

FIG. 7 is a schematic representation of reactions for illustrating theinvolvement of copper-zinc-thionein in the glutathione-peroxidase (GP)system.

FIG. 8 is a graph for determining the Km value for the action of the MTon the GP system.

FIG. 9 is a graph illustrating the site of action of the MT on the GPsystem.

WORKING EXAMPLES

The pharmacological activities of several antioxidative biophylacticagent according to the invention are illustrated in the followingexamples.

I. ANTIOXIDATIVE BIOPHYLACTIC ACTIONS WHICH MT-INDUCING SUBSTANCES EXERT

Since, as mentioned above, the mechanism of living body damaging due tooxidative invasions and the protective mechanism are fundamentallycommon in large part irrespective of the damage-causing factor, aparticular case of radiation damaging is given herein as a typicalexample.

TEST EXAMPLE 1 (EFFECTS OF VARIOUS METALS)

Manganese (Mn), cadmium (Cd) and zinc (Zn) were administered to micebeforehand and the whole body each mouse was then irradiated with Xrays. The body weight changes and mortality rates were recorded and theliver MT levels were determined.

(1) Method

The animals used were ddY-strain clean male mice (7 weeks of age). MnCl₂(10 mg Mn/kg body weight), CdCl₂ (3 mg Cd/kg body weight) or Zn(CH₃COO)₂ (20 mg Zn/kg body weight) was dissolved in 0.9% aqueous sodiumchloride solution and given to the mice by subcutaneous injection eachat a dose not higher than the semilethal dose. After 24 hours, the micewere irradiated with X rays at a dose of 630 rad almost corresponding tothe LD₅₀. Thereafter, the body weight changes in the mice and themortality rates were recorded for 30 days. At the same time, the mouseliver was excised 24 hours after administration of those metals, namelyimmediately before X ray irradiation, and 24 hours after X rayirradiation and the MT content in each liver homogenate was determinedby the Cd hemoglobin saturation method of Onosaka et al. [Eisei Kagaku(Hygienic Chemistry), 24, 128-131 (1978)].

(2) Results

The cumulative mortalities in mice of the respective groups afterirradiation with 630 rad of X rays are shown in FIG. 1. In all themetal-dosed groups, namely the Mn, Cd and Zn groups, reductions inradiation-induced mortality were found as compared with the control(undosed) group. In each of the Cd- and Mn-dosed groups, in particular,the cumulative mortality was 0.06, namely not higher than one tenth ofthat in the control group, namely 0.75. It was thus confirmed thatpreliminary administration of Mn or Cd can reduce radiation damagesremarkably. The changes in mouse body weight as followed on thatoccasion for 30 days revealed a typical two peak body weight decreasecurve characteristic of radiation damages for the control group whereas,in the Mn-dosed group, the body weight decrease was almost negligiblebut, contrariwise, the body weight continued to increase even afterirradiation and the general condition of mice as evaluated on the basisof the appearnce of the coat of fur, the condition of anemia and thebody movement was obviously good. In the Cd-dosed group, typical bodyweight loss supposedly due to the toxicity of Cd itself was observed atthe first week of post irradiation but mice could survive and thegeneral condition was good as in the Mn-dosed group. On the other hand,individual differences in body weight change were great in the Zn-dosedgroup although a reduction in mortality was observed, as shown inFIG. 1. The fact that, in the case of Mn, a protective effect can beproduced at a dose not more than one twentieth of the semilethal dosebut that, in the case of Cd or Zn, a dose of about one half to one thirdof the semilethal dose is required may be associated with the abovefact.

The MT levels in the liver as determined 24 hours after the pretreatmenti.e. exactly before X-ray irradiation and 48 hours after thepretreatment i.e. 24 hours after irradiation in mice are shown in Table1.

                  TABLE 1    ______________________________________    MT concentrations in mouse liver (μg/g liver    tissue)                     Mean ± standard deviation                       Before ir- After ir-                       radiation  radiation                       (24 hours  (48 hours                       after      after    Pretreatment       pretreatment)                                  pretreatment)    ______________________________________    CdCl.sub.2             3     mg Cd/kg s.c.                               198 ± 41                                        563 ± 38    Zn-acetate             5     mg Zn/kg s.c.                               33 ± 5                                        88 ± 25    Zn-acetate             20    mg Zn/kg s.c.                               120 ± 18                                        189 ± 24    MnCl.sub.2             10    mg Mn/kg s.c.                               126 ± 16                                        156 ± 38    Physiological             5     mg/kg s.c.  25 ± 1                                        66 ± 14    saline    Undosed                    26 ± 1                                        71 ± 22    ______________________________________

About five- to seven-fold increases MT level in mouse liver immediatelybefore X ray irradiation were confirmed in the groups given 20 mg/kgbody weight of Zn, 3 mg/kg body weight of Cd and 10 mg/kg body weight ofMn, respectively, as compared with the control group level of 26±1 μg/gliver tissue. Furthermore, the mouse liver MT levels after X-rayirradiation show increases by not less than several tens of microgramsper gram as compared with the levels before irradiation in the dosedgroups as well as in the undosed group. Similarly, sub-cutaneousinjection of 1.25 mg/kg body weight of Cu resulted in an increase inmouse liver MT level to 118±21 μg/g liver tissue. In contrast to theseresults, salts incapable of MT induction, such as Mg salts, did not showany protective action against radiations.

TEST EXAMPLE 2 (EFFECTS OF THE DOSE OF Mn)

(1) Method

The procedure of Test Example 1 was followed except that the dose of Mnwas varied within the range of 5-50 mg Mn/kg and that the irradiationwas performed at a semilethal dose of 630 rad and at a lethal dose of720 rad.

(2) Results

The mortalities for the respective mouse groups after irradiation with720 rad of X rays are shown in FIG. 3 and the liver MT levels 24 hoursafter irradiation are shown in Table 2. It was confirmed that, as shownin Table 2, the liver MT level increases with the increase of the doseof Mn and that, as shown in FIG. 3, the survival rate increases as wellwith the increase of the dose of Mn within the range of 5-20 mg Mn/kg.The decrease of the survival rate in the case of 50 mg Mn/kg issupposedly due to the toxicity of Mn itself.

                  TABLE 2    ______________________________________    MT formation versus dose of Mn    Dose of Mn   Liver MT concentration    (mg Mn/kg)   (nmol/g tissue)    ______________________________________    0       (control)                      3.01 ± 0.08    5                 4.55 ± 0.80    10               13.30 ± 2.82    20               24.38 ± 6.61    50               29.13 ± 2.70    ______________________________________

The relation between the survival rate in each group of mice afterirradiation with 630 rad of X rays and the dose of Mn and the relationbetween said rate and the logarithm of the liver MT concentration areshown in FIG. 4A and FIG. 4B, respectively, with the coefficients ofcorrelation as found by simple regression analysis. The results indicatethat the mouse survival rate is directly correlated with the MTconcentration in the liver rather than with the dose of Mn.

TEST EXAMPLE 3 (EFFECT OF Zn VERSUS RADIATION DOSE)

Water containing the Zn ion in a high concentration (1,000 ppm) waspreadministered to mice, the mice were then subjected to whole-bodyirradiation with gamma rays, and the biophylactic effect versus theradiation dose was investigated.

FIG. 5 shows the cumulative mortality versus the number of days afterirradiation of mice given Zn-containing drinking water with gamma raysat a semilethal or lethal dose. A decrease in mortality and a life spanprolonging effect were observed in the Zn-drinking group as comparedwith the control group. The results also indicate that the effect ofirradiation is not weakened when the radiation dose is large and equalsor exceeds the lethal dose but that a biophylactic effect is producedwhen the radiation dose is lower.

TEST EXAMPLE 4 (MT INDUCTION BY HEMOLYTIC STREPTOCOCCI AND BY A CORIOLUSVERSICOLOR EXTRACT)

OK-432 (Chugai Pharmaceutical Co.; trademark Picibanil) and PS-K (SankyoPharmaceutical Co.-Kureha Chemical Industry Co.; trademark Krestin) wererespectively administered to mice and liver MT concentrations weredetermined. For comparison, MT-inducing metals such as cadmium (Cd),zinc (Zn) and manganese (Mn) were also administered.

(1) Method

The animals used were Jcl:ICR-strain male mice (7 weeks old and 9 weeksold). They had free access to diet and water and were fed in aconstant-temperature environment. The agents shown in Table 1 wereadministered either singly or continuously at one- or two-day intervalsin the dosage and manner indicated in the table and, 20 hours later, theliver MT levels were determined. The assay of MTs was performed by theCd-hemoglobin saturation method of Onozaki et al. [Eisei Kagaku, 24,128-131 (1978)] and by the Ag-hemoglobin saturation method of Cherian etal. [Toxicol., 23, 11-20 (1982)].

(2) Results

While the mouse liver MT concentration in untreated mice of 4 to 25weeks of age generally amounts to about 2-3 nmol/g, OR-432 and PS-K, inboth the cases of single administration and prolonged administration,gave values several times higher as compared with mice in the controlgroup and thus each showed a high MT induction capacity comparable tothat found in metal administration.

                                      TABLE 3    __________________________________________________________________________    NT formation in the mouse liver after various kinds of pretreatment                                     Liver MT concentration                              Age of mice                                     (nmol/g tissue)              Pretreatment    (weeks)                                     Cd-hem method                                             Ag-hem method    __________________________________________________________________________    OK-432    10                KE/animal × once, i.p.                              7      14 ± 5                                              13 ± 5.6              1 KE/aminal × 10 times, i.p.                              9      19 ± 11    PS-K      50                mg/kg × once, i.p.                              7      5.7 ± 1.1              50                mg/kg × 9 times, i.p.                              9       22 ± 9.3    CdCl.sub.2              3 mg Cd/kg × once, s.c.                              7       25 ± 5.0    Zn-acetate              20                mg Zn/kg × once, s.c.                              7       15 ± 2.3    MnCl.sub.2              10                mg Mn/kg × once, s.c.                              7       16 ± 2.0    physiological saline              6.7                ml/kg × once, i.p.                              7      2.8 ± 1.0                                             1.7 ± 0.2              6.7                ml/kg × 10 times, i.p.                              9      1.5 ± 0.4    __________________________________________________________________________     Each result given is the mean of 5 animals, accompanied by the standard     deviation.

TEST EXAMPLE 5 (PROTECTIVE ACTION OF HEMOLYTIC STREPTOCOCCI AGAINSTIRRADIATION)

OK-432 (Chugai Pharmaceutical Co.; trademark Picibanil) waspreadministered to mice, whole-body X ray irradiation was thenconducted, and the survival rate after irradiation was recorded. Itsactivity was compared with that of manganese (Mn).

(1) Method

The animals used were Jcl:ICR-strain male mice (7 weeks of age). OR-432(5 KE/animal) and MnCl₂ (20 mg Mn/kg body weight) were respectivelydissolved in 0.9% aqueous sodium chloride solution, and OK-432 was givenby intraperitoneal injection and MnCl₂ by subcutaneous injection. Oneday later, the dosed groups, together with a control group, wereirradiated with the lethal dose of 720 rad of X rays, and the survivalrates were recorded for the subsequent 30 days.

(2) Results

The survival rates of the respective groups of mice after irradiationwith 720 rad of X rays are shown in FIG. 6. The data confirmed thatreductions in the rate of deaths due to irradiation as compared with thecontrol (undosed) group are found in the OK-432 and Mn groups and that aradiation damage reducing activity which is comparable to that of Mnpreadministration can be obtained by preadministration of OK-432.

II. ACTION IN THE ANTIOXIDATIVE BIOPHYLAXIS MECHANISM IN WHICH MTs AREINVOLVED

As already mentioned hereinabove, the glutathione (GHS)-peroxidasesystem (GP system) plays an important role in the antioxidativebiophylaxis mechanism in living organisms. In the GP system shown inFIG. 7, GSH-peroxidase (GP) reduces peroxides. For the reduction, GSH isrequired as an electron donor, and, as a result, GSH is converted tooxidized-form glutathione (GSSG). Since the oxidized GSH, namely GSSG,is apt to flow out of the cell, it is naturally conceivable thatintracellular GSH might sometimes become insufficient in quantity Infact, it has been observed that the susceptibility to radiationsincreases when the GSH level is decreased [Ohara et al.: Exp. Cell.Res., 58, 182-185 (1970); Modig et al.: Int. J. Radiat. Biol., 22,258-268 (1971); etc.]. It has also been reported that a reduction in GSHlevel leads to reduced resistance to radiations upon exposure thereto aswell as to various pathological phenomena such as ulceration in thestomach, canceration of cells and hemolysis [Boyd et al.: Science, 205,1010-1012 (1979); etc.].

The inventor confirmed that, like GSH, MTs can work as the alternativeof GSH to react with GP. This is illustrated in detail in the following.

(1) Method

To examine whether MTs can serve as electron donors to the GP system,namely as substitutes for GSH, under conditions in which GSH is absent,an in vitro system shown in FIG. 7 was constructed. Thus, the reductionof the substrate t-butyl-OOH by GP was put in the coupled NADPHoxidation system, as shown in FIG. 7, and the change of absorbance at340 nm as caused by consumption of NADPH in the reaction mixture in acuvette was followed using a double-beam spectrophotometer. Theprocedure was as follows: Two cuvettes for the spectrophotometer arefilled with the following solution in exactly the same volume. Thereaction mixture solution is principally 250 mM (hereinafter eachconcentraiton being a final concentration) phosphate buffer (pH 7.4)with NaN₃ added to a concentration of 2.5 mM and further supplementedwith 0.3 unit/ml each of GP and glutathione reductase (GR), 5 mM t-butylOOH and 0.5 mM NADPH, for both cuvettes. Finally, GSH or MT is added toone of the cuvettes, when the reaction starts, and the difference inabsorbance between the cuvettes is observed of a recorder. In this way,the decrease in the quantity of NADPH per unit time (rate of reaction)is observed. By increasing the concentration of GSH or MT stepwise(titration), the Km value (Michaelis constant) for the enzymaticreaction of GP can be determined.

(2) Results

FIG. 8 shows the results of the Km value measurement for MT to GP bystepwise addition of MT, as alternative to GSH, to the GP system. It wasrepeatedly confirmed that the Km value for MT as determined on the basisof the Lineweaver-Burke plots shown in the figure is in the order of 0.1mM. On the other hand, the Km value for GSH was about 4 mM. The MT usedwas copper-zinc-thionein purified from the calf liver. When, in thereaction shown in FIG. 7, GP, GR and NADPH are first added to thereaction mixture and then MT is added, no reaction is started. On thecontrary, when t-butyl-OOH is added thereto, the reaction starts, asshown in FIG. 9. This indicates that MT serves as an electron donor tothe GP system, not to GR.

While the MT level in cells may vary over a wide range of 7-500 μg/g wettissue, the liver MT level in normal mice, when measured by theinventor, differed little according to sex or age (in weeks) and wasabout 20 μg/g, that is about 3 μM, but increased to 120 to hundreds ofmicrograms per gram (20-100 μM) upon stress loading such as metaladministration. The liver MT level in human is higher than in animalsand, according to a report of Onozaka et al., it is 200-550 μg/g[Onozaka et al.: Eisei Kagaku, 30, 173-176 (1984)]. The Km value forcopper-zinc-thionein to GP as determined by the inventor is about 100 μMand therefore this reaction is considered to be able to proceedphysiologically when MTs are sufficiently induced and synthesized inliving organisms.

INDUSTRIAL APPLICABILITY

As described hereinabove, the antioxidative biophylactic agent accordingto the invention induces metallothioneins (MTs) in living organisms andthese MTs cause the biophylactic function to be performed in lieu ofglutathione (GSH). Therefore, said agent does not inhibit free radicalformation or eliminate free radicals in a direct manner upon oxidativeinvasions such as irradiation but prevent various functional disordersdue to biomembrane damages and increases the resistance and recovery ofcells. In particular, manganese and compounds thereof, hemolyticstreptococci and Coriolus versicolor extracts are remarkably effective.Among others, OK-432 and PS-K have already been applied to human bodiesas antitumor agents and accordingly their safety has been confirmed.Therefore, in cases where the possibility of exposure to radiations willincrease in the future, for example in the case of workers in thenuclear power industry, astronauts working in the cosmic field, workersin the medical field who are engaged in diagnosis and treatment of suchdiseases as cancer, or patients who have to be exposed to undesirableirradiation for the purpose of diagnosis, the biophylactic function inhuman bodies can be promoted by administering said agent in advance as aprophylactic means of preventing possible radiation damages.

What is claimed is:
 1. A method of protecting a host from radiationdamage, comprising administering to a host in need thereof an effectiveradiation damage protecting amount of a water-soluble manganese salt inan amount effective to increase the liver metallothionein level in thehost by at least fivefold within about one day following theadministration.
 2. The method of claim 1, wherein the water-soluble saltis administered to the host at about 5-30 mg manganese per kg bodyweight of the host.
 3. The method of claim 1, wherein the water-solublesalt is administered to the host at about 10-20 mg manganese per kg bodyweight of the host.
 4. The method of claim 1, wherein the water-solublemanganese salt is administered to the host orally or by injection.